13q Deletion Syndrome Involving RB1: Characterization of a New Minimal Critical Region for Psychomotor Delay

Retinoblastoma (RB) is an ocular tumor of the pediatric age caused by biallelic inactivation of the RB1 gene (13q14). About 10% of cases are due to gross-sized molecular deletions. The deletions can involve the surrounding genes delineating a contiguous gene syndrome characterized by RB, developmental anomalies, and peculiar facial dysmorphisms. Overlapping deletions previously found by traditional and/or molecular cytogenetic analysis allowed to define some critical regions for intellectual disability (ID) and multiple congenital anomalies, with key candidate genes. In the present study, using array-CGH, we characterized seven new patients with interstitial 13q deletion involving RB1. Among these cases, three patients with medium or large 13q deletions did not present psychomotor delay. This allowed defining a minimal critical region for ID that excludes the previously suggested candidate genes (HTR2A, NUFIP1, PCDH8, and PCDH17). The region contains 36 genes including NBEA, which emerged as the candidate gene associated with developmental delay. In addition, MAB21L1, DCLK1, EXOSC8, and SPART haploinsufficiency might contribute to the observed impaired neurodevelopmental phenotype. In conclusion, this study adds important novelties to the 13q deletion syndrome, although further studies are needed to better characterize the contribution of different genes and to understand how the haploinsufficiency of this region can determine ID.


Introduction
Retinoblastoma (RB) is the most frequent intraocular tumor of the pediatric age with an incidence of 1/15,000-28,000 live births [1]. It is caused by biallelic inactivation of the RB1 oncosuppressor gene located in 13q14.2 [1]. Constitutive gross-sized deletions involving RB1 are present in 10% of cases [2][3][4][5]. When the deletion involves part of the RB1 surrounding genome, it causes a rare contiguous gene deletion condition known as 13q deletion syndrome [1]. The syndrome is characterized by RB, developmental anomalies, and peculiar facial features.
In 1983 the facial phenotype of patients with RB and 13q deletions was firstly described, observing three Japanese cases with prominent eyebrows, a broad nasal bridge, a bulbous tip of the nose, a large mouth with a thin upper lip, and a long prominent philtrum [6]. In a subsequent paper, Baud et al. [7] described 22 RB patients showing anteverted ear lobes, a high and broad forehead, a short nose, and a thick everted lower lip. Depending on the size and location of the deleted region, patients showed severe intellectual disability (ID) and/or motor impairment. In 2001, Bojinova et al. [8] described an additional 13 patients with cranial anomalies, frontal bossing, broad cheeks, and large ears. Additional case reports of 13q deleted patients involving band 13q14 reported macrocephaly, hypertelorism, proptosis, cleft palate, macroglossia, hypotonia, and mild to severe developmental delay [9,10]. In 2007, Caselli et al. [1], by array-CGH, identified two deleted patients who showed variable clinical features including craniofacial dysmorphism, psychomotor delay, hypotonia, short stature, and anomalies of feet and brain. In 2011, Mitter and colleagues [11] collected and reported clinical, cytogenetic, and molecular data of 63 patients with isolated or familial RB who carried an interstitial 13q deletion involving RB1. Patients with a small deletion (within 13q14 and smaller than 6 Mb) displayed macrocephaly, tall stature, obesity, motor, and/or speech delay [11]. Patients with a medium deletion (within 13q12.3q21.2 and sized between 6-20 Mb) showed characteristic facial features, mild to moderate psychomotor delay, short stature, and microcephaly [11]. Patients with a large deletion (within 13q12q31.2 and larger than 20 Mb) had characteristic craniofacial dysmorphisms, mild to severe psychomotor delay, hypotonia, constipation, and feeding problems. Additional features included deafness, seizures, and brain and heart anomalies [11].
13q deletion syndrome involving RB1 is a rare contiguous gene deletion condition whose genotype-phenotype correlations are still a question of debate. In order to explain ID, Caselli et al. [1] suggested a critical region in 13q14, containing at least 30 genes, among which four genes were considered as good functional candidates for neurodevelopmental delay: NUFIP1 (nuclear fragile X mental retardation protein 1), HTR2A (serotonin receptor 2A), PCDH8 (protocadherin 8), and PCDH17 (protocadherin 17). Mitter et al. [11] suggested that heterozygous loss of NUFIP1 and PCDH8 may contribute to psychomotor delay, deletion of MTLR1 to microcephaly, and loss of EDNRB to feeding difficulties and deafness [11].
In the present study, we describe seven new patients with 13q deletion involving RB1 and discuss genotype-phenotype correlation. Array-CGH analysis was performed in all patients, and results showed medium and large deletions of the 13q chromosome, ranging from 13q13.2 to 13q22.3. Overlapping of the deleted regions allows furthering the current knowledge, identifying a new minimal critical region for psychomotor delay, and excluding previously suggested candidate genes.

Human Subjects
Patients came to the Medical Genetics department after been evaluated and hospitalized by the Unit of Ophthalmology and Retinoblastoma Referral Center, and the Unit of Pediatrics, Department of Maternal, Newborn and Child Health (University of Siena, Policlinico "Santa Maria alle Scotte"). For each patient genetic counseling was performed in order to collect family history and better define facial and physical characteristics. All patients' parents gave their written informed consent to the study that was carried out according to the Declaration of Helsinki.
Genomic DNA was extracted from EDTA peripheral blood samples using MagCore HF16 (Diatech Lab Line), according to the manufacturer's instructions. The DNA quantity was estimated using the NanoDropTM 2000/2000c Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).

Whole-Genome Array-Based Comparative Genomic (a-CGH)
High-resolution whole-genome array-based Comparative Genomic Hybridization (a-CGH) analysis was performed on genomic DNA of the patients and their parents, using the SurePrint G3 Human CGH Microarray 8 × 60k (Agilent Technologies, Santa Clara, CA, USA), a dual-color array containing 60-mer high-quality probes with 41 Kb genome-wide median probe spacing. Copy Number Variants (CNVs) were analyzed and mapped using the Human Genome Assembly GRCh37/hg19. Slides were scanned using an Agilent G2565CA Microarray Scanner (Agilent Technologies, Santa Clara, CA, USA) and processed using Feature Extraction software (v10. 5

Clinical Description
Clinical findings of each patient are described in Tables 1 and 2. Diagnosis of RB was performed in all patients within the first year of life, except in Patient 2 who received an RB diagnosis at 3 years of age. Three patients did not show developmental delay over time (as confirmed by subsequent follow-up). Dysmorphic features were observed except for Patient 1 (Tables 1 and 2).

Patient 1 (#349/19)
We first visited patient 1 when she was 4 years old (Table 1). She was the second child of healthy unrelated parents. Family history was silent for RB or other types of tumors. She was born from spontaneous delivery at 30 weeks of gestation, due to premature rupture of membranes. Birth weight was 1.5 kg (50 • -75 • percentile), length was 46 cm (>90 • percentile) ( Table 1); for the first month of life, the baby required assistance and remained in the incubator, whereas valid suction was reported (Table 1). At five months, unilateral right RB was diagnosed, firstly treated with chemotherapy and laser therapy with a reduction of the mass. Then, because of the tumor's relapse, she again performed chemotherapy and laser therapy and finally enucleation ( Table 1). The contralateral eye's controls were always normal. Brain MRI revealed the presence of frontal capillary angioma ( Table 2). Normal psychomotor development was reported with first words at 1 year and independent walking between 12 and 18 months (     In the context of prenatal diagnostic testing, the karyotype identified a pericentric inversion of chromosome 9, inv(9) (p11q13). Prenatal and postnatal periods were normal. Auxological parameters at birth were not available. Normal psychomotor development was reported: babbling at 6-7 months and independent walking at 15 months (Table 1). At the time of the examination, the patient spoke Greek very well and understood enough Dutch. At 3 years and 6 months of age, bilateral RB was diagnosed involving predominantly the right eye, for which enucleation was required, and at 70% the left eye, treated with chemotherapy (Table 1). A physical examination, at 4 years old, showed a length of 103 cm (25 • -50 • percentile), a weight of 17 kg (50 • -75 • percentile), and OFC of 51cm (25 • -50 • percentile) ( Table 1). She presented prominent frontal bossing, broad and wide forehead, deeply set eyes, bilateral overfolded helix, and broad nasal bridge with hypoplastic nostrils ( Table 2). Clinicians who had taken care of the patient over time reported her last follow-up at 12 years of age without any important problems or learning difficulties, normal growing, and regular social life (Table 1).

Patient 3 (#2148/18)
We first visited patient 3 when she was 5 months old. She was born at term, without any problems during pregnancy. Parents reported previous recurrent miscarriages; however, their karyotype was normal. The growth parameters at birth were in the high normal range (length 52 cm, 85 • -97 • percentile; weight 4.0 kg, 85 • -97 • percentile; and OFC 38 cm, 95 • percentile) ( Table 1). At 4 months, unilateral left RB was diagnosed, and enucleation was needed (Table 1). A physical examination, at 1 year and 5 months, showed a weight of 11.5 kg (25 • percentile), and OFC of 51cm (>99 • percentile) ( Table 1). She presented a wide and broad forehead, prominent frontal bossing, bushy eyebrows in the third medium and sparse in the lateral third, hypertelorism, deeply set eyes, depressed nasal root, broad nasal tip, everted nostrils, prominent filter, Cupid's bow, and everted lower lip ( Table 2). The patient walked independently, acquired fine motor skills, and pronounced a few words (Table 1). Clinicians reported that at the age of three the patient used good language, while sphincter control was not yet acquired (Table 1).

Patient 4 (#2794/20)
Patient 4 was a 9-month-old female, the second of healthy and unrelated parents ( Table 1). The family history was negative for other cases of RB and other ocular problems. On the paternal side, there were many relatives with different types of tumors. She was born at 39 weeks of gestation by cesarean section, because of maternal scoliosis. Pregnancy was complicated by hypertension and intrauterine growth retardation, in absence of flowmetry alterations. Growth parameters at birth were: length 47 cm (15 • percentile); weight 2.25 kg (<5 • percentile) ( Table 1). Hypovalid suction and reflux were reported during the perinatal period. At 9 months, unilateral right RB was diagnosed and chemotherapy was required (Table 1).
A physical examination at nine months showed a length of 63 cm (<5 • percentile), a weight of 6.63 kg (75 • -90 • percentile), and OFC of 38.8 cm (<5 • percentile). No sitting position was acquired; the patient presented bushy eyebrows, wide forehead, small eyes with apparent hypertelorism, broad nasal bridge, long and prominent filter, low-set posteriorly rotated ears (Table 2).
At 1 year and 3 months, the patient showed important neuromotor delay (Table 1). She acquired sitting position and fine motor skills; babbling was absent, no crawling or walking movements were observed. Highlighted only supine to prone rolling (Table 1).
At 7 years old, both showed an important psychomotor development delay: language was not still acquired, nor independent walking or sphincter control (Table 1). They both showed important stereotyped hand movements along the midline and movements of the trunk (Table 1).
At 13 years old, both had not yet acquired any language skills, nor independent walking. Hands stereotypes were no longer present; their sexual development was normal (Table 1). 3.1.6. Patient 7 (#3311/17) Patient 7 was an 8-month-old male, the second child of healthy and unrelated parents. The family history was silent for ocular problems and tumors. He was born at 36 weeks of gestation; his pregnancy was complicated by oligohydramnios during the last trimester. Growth parameters at birth were: length 45 cm (>25 • percentile), weight 2.095 kg (10 • percentile), and OFC 32.9 cm (50 • percentile) ( Table 1). Absent gaze and strabismus were noticed since the first days of life. A neurological examination reported: reduced motor findings, axial hypotonus, poor reactivity to auditory stimuli. The V.E.P (Visual Evoked Potential) found possible bilateral sensorineural hearing loss.
At 7 months, bilateral RB was diagnosed, then treated with chemotherapy (Table 1). Brain-MRI showed "Plagiocephaly and hypoplastic brain stem". Growth parameters at 8 months were in the normal range (length 60 cm, 50 • percentile; weight 5.8 kg, 25 • percentile (Table 1). He presented global developmental delay and hypotonia, poor head control, absent babbling (Table 1). Facial features included frontal bossing, wide and broad forehead, hypertrichosis, bushy eyebrows in the lateral third, low-set ears, depressed nasal root, prominent filter, slight micrognathia, and prominent lower lip (Table 2).
At 2 years old, clinicians reported axial hypotonia, poor head control, and motor skills. Sitting position was not acquired, and language was still absent; it was reported only liquid feeding because of chewing difficulties (Table 1).

Molecular Characterization
Results obtained by Array-CGH are summarized in Table 3; Copy Number Variants (CNVs) breakpoints all refer to the Human Genome Assembly GRCh37/hg19. The analysis revealed medium and large deletions of the 13q chromosome, spanning from 13q12.3 to 13q22.3. Segregation analysis in parents of all patients except Patient 2 showed the de novo origin of each deletion. The analysis did not identify any additional CNV in the whole cohort. In order to identify a minimal critical region for developmental delay, we excluded from our analysis the regions found in Patients 1,2,3, who did not show global developmental delay or ID, and we overlapped the rearrangements of the remaining patients. The study allowed us to focus on cytobands 13q13.3q14.11 (Figure 1 (Figure 1). Among these, five are genes already reported to play a role in ID (Table 4).  Table 4. Genes reported to play a role in ID.

Gene Symbol (OMIM#). Phenotype (OMIM#) Inheritance Association with Neurodevelopmental Disorders
NBEA (OMIM#604889) Neurodevelopmental disorder with or without early-onset generalized epilepsy (OMIM#619157) AD Reported mutated in autosomal dominant neurodevelopmental disorder with or without epilepsy [12]. It was cited with a score of 1 in the SFARI Gene database as a candidate gene for autism spectrum disorder [13]. It is involved in synaptic transmission and exocytosis [14], neuron excitability, and brain development [15].
Cerebellar, ocular, craniofacial, and genital syndrome (OMIM#618479) AR Loss-of-function mutations cause an extremely rare autosomal recessive condition, the Cerebellar, ocular, craniofacial, and genital (COFG) syndrome, characterized by moderate to severe developmental delay and impaired intellectual development [16].

Discussion
Patients with a 13q deletion containing RB1 show a variable phenotype that, depending on the size and location of the deleted region, includes ID, growth retardation, craniofacial dysmorphisms, congenital malformations, and increased risk of retinoblastoma. In the present study, we characterized by array-CGH seven new cases with 13q deletion involving RB1 and discussed genotype-phenotype correlations.
Among the deleted genes, five of them were selected because of their involvement in impaired neurogenesis, when mutated (Table 4). In particular, NBEA encodes for Neurobeachin, a brain-specific kinase-anchoring protein implicated in vesicle trafficking and synaptic structure and function [12][13][14][15]. The gene is reported mutated in neurodevelopmental disorder with or without epilepsy [16] and it was cited with a score of 1 in the SFARI Gene database as a candidate gene for autism spectrum disorder (data referred to the latest SFARI Gene database update, June 20th, 2019) [17]. Studies on the murin Nbea gene, which shares high homology to human NBEA [18], established the involvement of Nbea in synaptic transmission and exocytosis [19], whereas other animal studies suggested a role in neuronal excitability [20] and brain development [21].
Concerning the remaining genes, MAB21L1, DCLK1, EXOSC8, and SPART haploinsufficiency might contribute to the observed psychomotor delay. MAB21L1 is contained within the neurobeachin gene [22] and belongs to the conserved male abnormal gene family 21 (Mab21) [23]. Mab21 was first described in the nematode Caenorhabditis elegans as a transcription factor in cell fate determination [24]. Mab21 genes play a major role in embryonic development, but gene expression extends beyond the developmental period well into adulthood [25]. Biallelic MAB21L1 loss-of-function mutations cause an extremely rare autosomal recessive recognizable condition, the Cerebellar, Ocular, cranioFacial, and Genital syndrome (COFG syndrome), which could be reported in association with moderate-tosevere developmental delay/ID and behavioral abnormalities [23]. DCLK1 was originally identified as a causative gene of cerebral cortical malformation such as lissencephaly [26,27] and was later found to regulate neuronal migration and axon outgrowth [26][27][28][29][30][31]. DCLK1 exerts multiple roles in neurogenesis, neuronal migration, axon/dendrite growth, and spine formation [32][33][34][35]. Recent genome-wide association studies (GWAS) and transcriptome analysis indicate that the DCLK1 gene might be a candidate gene for neurodevelopmental and neuropsychiatric disorders [36,37]. EXOSC8 is a member of the RNA exosome, a conserved multi-protein complex that is essential for correct RNA processing and degradation [38,39]. Recessive variants in exosome components EXOSC3, EXOSC8, and RBM7 cause various constellations of pontocerebellar hypoplasia (PCH), spinal muscular atrophy (SMA), and central nervous system hypomyelination [40,41]. Exosomal proteins are important not only for neuronal development but also for the survival of spinal motor and cerebellar neurons [42]. SPART is the pathogenic gene causing Troyer Syndrome, an extremely rare autosomal recessive disease that affects the nervous system, characterized by spastic paraplegia and distal amyotrophy [43,44]. Spartin has different expression levels in various tissues and organs, and it is highly expressed in the temporal lobe, anterior central gyrus, corpus callosum, adipose tissue, and the placenta [45,46].
Concerning craniofacial dysmorphisms, in our group of patients, a broad and wide forehead was reported in 85.7% (6/7); a prominent filter in 71.4% (5/7); ear abnormalities (everted or folded helix, low-set/everted earlobes), frontal bossing, bushy eyebrows, deepset or small eyes, thick everted lower lip, brain anomalies, and altered head morphology in 43% (3/7); depressed nasal root, everted or hypoplastic nostrils, broad nasal bridge, clinodactyly, and cutis marmorata in 28.5% (2/7). Among other less common features, hypertrichosis, micrognathia, cardiac defects, and hearing loss were observed in 14.2% of the cases (1/7). With the exception of Patient 1, who did not show any particular trait, our data overall confirm a previous correlation between facial dysmorphisms/congenital anomalies and the 13q12.3q31 region, identifying it as a critical region related to these broad phenotypic spectra [11].
In conclusion, this study adds novelties regarding the genotype-phenotype correlation in 13q deletion syndrome including RB1, defining a new key region for developmental delay. The focus on the 13q13.3q14.11 region allowed us to exclude the possible involvement of the previously reported candidate genes HTR2A, NUFIP1, PCDH8, and PCDH17 in ID and global psychomotor delay development [1]. Further studies are however needed to characterize the contribution of different genes involved and to understand the impact of the haploinsufficiency of this region on ID. Moreover, as more patients with overlapping deletion will be identified, more precise conclusions on genotype/phenotype correlation can be drawn.
It is well ascertained that neurodevelopmental disorders can arise from many different genetic abnormalities ranging from chromosomal and genomic defects to single nucleotide variations in single genes. The array-CGH performed in our case series did not identify any additional CNV possibly accounting for the neurodevelopmental phenotype of our patients. However, it cannot be ruled out that smaller CNVs or single-nucleotide variations undetectable by array-CGH may act as cofactors for ID.

Conclusions
The study provides new clinical and molecular information useful for the management of cases with a 13q deletion, especially in the prenatal and neonatal settings. A better understanding of the genotype/phenotype correlation can enable clinical geneticists to provide more definite prognostic information to couples who receive a prenatal diagnosis of 13q deletion. Furthermore, our data reinforce the idea that once 13q deletion syndrome diagnosis is established, a multidisciplinary approach is needed by a team of specialists, including, besides the ophthalmologist, pediatric oncologist, pathologist, and radiation oncologist, also the neuropsychiatrist, the speech therapist, and the psychomotor therapist, in order to fully manage all the different aspects of the syndrome and their progression over time

Institutional Review Board Statement:
The study is part of a larger study entitled: "Sviluppo di nuove strategie per identificare marcatori prognostici e predittivi di risposta alla terapia nel paziente pediatrico affetto da retinoblastoma", protocol code "LIQUID-EYE RETINO", approved on 2 July 2020. Data Availability Statement: Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.